Hepatitis E virus (HEV) is an important but extremely understudied pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 infect humans and other animals. The long-term goals of this project are to delineate the mechanisms of HEV cross-species infection and identify host and viral determinants of host range and pathogenesis. In specific aim 1, we hypothesize that the hypervariable region (HVR) in ORF1 and the inserted host gene sequences in HVR determine HEV host range. Our preliminary studies suggested that HVR and its adjacent region are involved in HEV tropism. The recent findings that host S17 and S19 gene sequences were inserted into the HVR of HEV that resulted in recombinant viruses with expanded cell tropism of different animal origins lends further credence to our hypothesis. We will determine (a) if a genotype 1 human HEV (infecting humans only) will acquire the ability to infect pigs and rabbits if its HVR is replaced with that o a zoonotic genotype 3 HEV, (b) if the genotype 1 human HEV with an inserted host S17 sequence in the HVR will acquire the ability to infect pigs and rabbits, and (c) if the adjacent X domain in ORF1 plays a role, together with HVR, in determining the host range. In specific aim 2, we hypothesize that the genotype- specific HVR sequences and/or inserted host sequences contribute to HEV pathogenicity. Increased pathogenicity by recombination events between host gene sequence and virus genomes has been reported for other viruses. The fact that the neurological and chronic cases of hepatitis E are all caused by the zoonotic genotype 3 HEV and that quasispecies compartmentalization in CSF and serum of clonal HEV sequences within the HVR region was identified from neurological cases suggested that the HVR plays a role in virus pathogenicity and emergence of neurotropic variants. We will determine (a) if the genotype 3 virus with the S17 insertion in HVR is more virulent than the virus without the insertion, (b) if the genotype 3 virus with a S17 insertion establishes chronic infection in pigs, and (c) if the HVR and its adjacent regions evolve during chronic HEV infection in pigs to produce neurotropic variants. In specific aim 3, we hypothesize that interferon stimulated gene 15 (ISG15) plays a role in HEV infection and host anti-HEV response. Our preliminary studies showed that HEV replication requires an active ubiquitin-proteasome system and that overexpression of ISG15 inhibited HEV replication in a HEV replicon system. The findings that the papain-like cysteine protease (PCP) domain in HEV ORF1 has deubiquitinating activity and carries out deISGylation of ISG15-conjugated proteins support our hypothesis. By using an efficient HEV infection cell culture model with genotype 3 HEV Kernow C- 1 strain, we will determine (a) if overexpression of ISG15 will inhibit genotype 3 HEV infection, and if so, what is the mechanism of inhibition, (b) if HEV counteracts ISG15 production and ISGylation, and if so, if the PCP domain plays a role in antagonizing ISG15 function. By completing this project, we expect to identify the viral determinant(s) for HEV host range and pathogenicity and delineate the role of ISG15 in anti-HEV response.